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Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado . Corresponding Author: Andrew Thorburn , Department of Pharmacology, University of Colorado School of Medicine, 12801 E. 17th Avenue, MS 8303, Aurora, CO 80045. Phone: 303-724-3290; Fax: 303-724-3664; E-mail: Andrew.Thorburn@ucdenver.edu doi: 10.1158/2159-8290.CD-17-0996 ©2017 American Association for Cancer Research.

IN THE SPOTLIGHT

Targeting the Lysosome for Cancer Therapy Christina G. Towers and Andrew Thorburn

Summary: Lysosomes are the recycling centers of the cell where organelles and proteins are degraded during autophagy and macropinocytosis; they also serve as signaling hubs that control the activity of mTORC1. In this issue, Rebecca and colleagues report the development of a new type of lysosomal inhibitor for cancer therapy that can inhibit multiple lysosomal activities that are needed for tumor cell survival and growth. Cancer Discov; 7(11); 1218–20. ©2017 AACR .

See related article by Rebecca et al., p. 1266 (8) .

Lysosomes are important for both catabolic pathways such as autophagy and macropinocytosis and anabolic growth pathways driven by mTORC1. These pathways are all potential targets in cancer. Autophagy is the process by which cellular material is delivered to the lysosome for deg-radation, and it has multiple roles in cancer ( 1 ). Although autophagy can have both protumor and antitumor effects, current efforts to deliberately target autophagy in cancer treatment focus primarily on trying to inhibit autophagy due to its ability to promote growth of established tumors and mediate chemoresistance during cancer therapy ( 2 ). Macropinocytosis is a specialized type of endocytosis that functions to deliver extracellular proteins to lysosomes; this promotes tumor growth, especially in RAS-driven tumors ( 3 ). mTOR is a critical regulator of cell growth and metabo-lism ( 4 ), and mTOR inhibitors have been widely tested as anticancer agents in preclinical models and patients ( 5 ). Antimalarial drugs such as chloroquine, hydroxychloro-quine, or quinacrine inhibit lysosomal function. However, these agents also have other effects and lower potency than we would like. Although chloroquine and hydroxychloro-quine are currently the only drugs that are used clinically as autophagy inhibitors and are being tested in dozens of trials ( 2 ), they do not affect other lysosomal activities, such as mTORC1 regulation, and they also have autophagy-independent effects as anticancer agents ( 6 ). For example, it has been known for many years that chloroquine can bind to DNA ( 7 ). A new article by Rebecca and colleagues ( 8 ) takes a systematic approach to lysosome inhibition and, for the fi rst time, describes an agent that can inhibit multiple cancer-driving functions of the lysosome.

In this new article, Rebecca and colleagues ( 8 ) extend a previous study from the same laboratories ( 9 ) that reported that dimerization of chloroquine could increase its potency

as an autophagy inhibitor by more systematically testing dimerized antimalarials. Dimerization of the oldest antima-larial drug, quinacrine, was particularly potent at suppress-ing tumor growth, and the authors went on to optimize the linker sequence. A systematic medicinal chemistry effort allowed them to achieve three important advances. First, they were able to make an agent that, unlike the parent anti-malarials, affects only the lysosome. Importantly, they also have versions that could not accumulate in lysosomes or inhibit lysosome function, and consequently have different biological effects as autophagy inducers versus inhibitors. Because these agents affect either lysosomes or DNA but not both, this allows more rigorous testing of the mechanism of action than is possible with previous lysosomal inhibitors like chloroquine. The second achievement focused on a lyso-some-specifi c agent called DQ661. The high potency and lys-osomal specifi city of the drug allowed DQ661 to be used as a photo-affi nity labeling probe to identify its molecular target as palmitoyl-protein thioesterase 1 (PPT1). PPT1 is required for depalmitoylation of proteins, and DQ661 has its effects on tumor cells by binding to and inhibiting this enzyme. A third advance showed that this drug has a very interesting multitarget activity profi le. It can block lysosomal activity and the major catabolic functions of autophagy and macro-pinocytosis, but can also inhibit mTORC1; this is the fi rst example of a molecule that can block both the signaling and degradative functions of lysosomes. Importantly, mTOR inhibition mediated by DQ661 involves a novel mechanism. DQ661 disrupts the complex that regulates mTORC1 at the lysosomal membrane. This ejects mTORC1 from the lysosome and prevents the amino acid–dependent regula-tion of mTORC1 kinase activity ( Fig. 1 ). Potential benefi ts of this different approach to mTOR inhibition are increased potency compared with catalytic inhibitors, broader activity against tumor cells with different genotypes and resistance mechanisms, increased functionality in nutrient-rich condi-tions, and the ability to cooperate with catalytic inhibitors of mTORC1.

The net effect of all these advances was that DQ661 was shown to have in vivo antitumor activity against different tumor models in both immunocompetent animals and immunodefi cient models. Importantly, the closely related drug DQ660, which does not target the lysosome but does cause tumor DNA damage, had little effect on tumor

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Figure 1.  DQ661 inhibits PPT1 to regulate multiple lysosome-mediated cellular processes. A, Under basal conditions in the absence of DQ661, the lysosome can fuse with both autophagosomes to mediate autophagic degradation and endosomes to recycle nutrients from the extracellular environment via macropinocytosis. In addition, the lysosome is the site of mTORC1-mediated signaling, which can result in decreased autophagosome initiation. B, DQ661 can bind directly to PPT1 in the lysosome, inhibiting recruitment of v-ATPase subunits and mTORC1 to the lysosomal membrane and disrupting the Ragula-tor complex and RHEB-mediated activation of mTORC1. This results in decreased protein synthesis, a buildup of autophagosomes that cannot fuse with the lysosome thus blocking autophagic flux, as well as a decrease in endosome fusion with lysosomes with a net decrease in proliferation, autophagic flux, and macropinocytosis. Inhibition of these multiple lysosomal processes results in an increase in apoptosis and decreased tumor growth.

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growth. This shows that the lysosome inhibition is critical for the antitumor effects of these molecules. Interestingly, in vivo activity was demonstrated in a tumor that is resist-ant to hydroxychloroquine and is not especially sensitive to genetic inhibition of either autophagy or mTOR alone. This is interesting because it has been argued that the key to success in targeting autophagy in cancer will be to focus on those tumors that are themselves especially autophagy dependent as defined by their high sensitivity to genetic interference with autophagy (2). However, the field currently lacks good biomarkers or oncogenic pro-files to identify autophagy-dependent tumors, and even tumor cells with driver mutations such as mutant RAS,

which has been associated with increased autophagy and sensitivity to autophagy inhibition (10), do not always respond to autophagy inhibition (11, 12). This raises the exciting notion that perhaps autophagy dependence may not be a necessary requirement for success when targeting autophagy using more potent multitargeting drugs like DQ661 and may broaden the applicability of autophagy inhibition as a therapeutic strategy.

As is often the case with important studies, the new find-ings also raise new questions. The authors’ data suggest that inhibition of PPT1 affects the proper localization of the lysosomal v-ATPase, which generates and maintains the pH gradient that keeps lysosomes acidic. But the effects

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observed with DQ661 must be more complex, as other drugs that block acidification are not able to block mTORC1 in the presence of amino acids. Thus, DQ661 may be a use-ful tool to further understand exactly how the mTORC1 complex is regulated at the lysosome membrane. Why does blocking protein depalmitoylation in the lysosome inhibit autophagy? Is this due to the effect on lysosomal pH, or are there other specific palmitoylated target proteins that are needed for autophagy? Will tumor cells be able to develop resistance to DQ661-mediated inhibition of mTOR as eas-ily as they circumvent the existing inhibitors? Why does interference with these processes kill tumor cells? And, will there be differences between tumors based on their suscepti-bility to inhibition of lysosome function with these agents? Finally, the identification of PPT1 as a new therapeutic tar-get in cancer suggests that protein palmitoylation deserves further investigation from the perspective of understand-ing both cancer biology and cancer treatment. This article opens the way to addressing these and other questions and to further testing of the efficacy of lysosome inhibition for cancer treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Grant SupportThe authors are supported by NIH grants RO1 CA150925, RO1

CA190170, R21 CA197887, and T32 CA190216.

Published online November 2, 2017.

RefeRenCeS 1. Amaravadi R, Kimmelman AC, White E. Recent insights into the

function of autophagy in cancer. Genes Dev 2016;30:1913–30. 2. Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer.

Nat Rev Cancer 2017;17:528–42. 3. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ,

Kamphorst JJ, Hackett S, et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 2013;497:633–7.

4. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell 2017;168:960–76.

5. Xie J, Wang X, Proud CG. mTOR inhibitors in cancer therapy. F1000Research 2016;5:2078.

6. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A. Chloroquine sensitizes breast cancer cells to chemo-therapy independent of autophagy. Autophagy 2012;8:200–12.

7. Kwakye-Berko F, Meshnick SR. Binding of chloroquine to DNA. Mol Biochem Parasitol 1989;35:51–5.

8. Rebecca VW, Nicastri MC, McLaughlin N, Fennelly C, McAfee Q, Ronghe A, et al. A unified approach to targeting the lysosome’s deg-radative and growth signaling roles. Cancer Discov 2017;7:1266–83.

9. McAfee Q, Zhang Z, Samanta A, Levi SM, Ma X-H, Piao S, et al. Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. Proc Natl Acad Sci U S A 2012;109:8253–8.

10. Guo JY, Chen H-Y, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011;25:460–70.

11. Eng CH, Wang Z, Tkach D, Toral-Barza L, Ugwonali S, Liu S, et al. Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy. Proc Natl Acad Sci U S A 2016;113:182–7.

12. Morgan MJ, Gamez G, Menke C, Hernandez A, Thorburn J, Gidan F, et al. Regulation of autophagy and chloroquine sensitivity by onco-genic RAS in vitro is context-dependent. Autophagy 2014;10:1814–26.

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